Summary Burkholderia pseudomallei (Bp) is a globally emerging Gram-negative pathogen and the etiologic agent of melioidosis, a multifaceted and difficult-to-treat disease. Our knowledge of the factors that allow Bp to resist antibacterials is still rudimentary. ?-lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. The chromosomally-encoded PenA Class A ?-lactamase is the single most important resistance determinant compromising clinical use of ?-lactam antibiotics with Bp. We discovered that PenA is a presumably outer membrane (OM)-associated lipoprotein, one of only three such ?-lactam lactamases described to date in Gram-negative bacteria. Membrane localization of these enzymes and its significance remains a mystery. We hypothesize that because OM membrane localization is not essential for enzyme activity or substrate specificity, it must serve another purpose. Our preliminary and recently published data with NDM-1 carbapenemase frame the overall hypothesis that PenA OM association facilitates its shedding in OM vesicles (OMVs), which can fuse with recipient cells to confer phenotypic rather than genetic resistance on susceptible recipient bacteria thus providing a niche population survival advantage in the presence of antibiotics. A role of a putative lipoprotein that is genetically closely linked to penA in this process cannot be excluded. To this hypothesis, we will build on our preliminary data and use the tools available in our laboratory to 1) investigate the biochemical properties of NlpD; 2) establish that PenA and NlpD are located in the OM and subsequently disseminated into OMVs; and 3) assess whether OMVs can fuse with recipient cells and bestow ?-lactam resistance and if so, whether fusion with recipient cells limited to kin. Studies of OM and OMV localization of clinically significant ?-lactamases contribute new knowledge in terms of antibiotic resistance, its diagnosis and therapeutic development.